Endosomes segregate endocytosed macromolecules destined to be degraded in the lysosome from molecules that are either recycled back to the cell surface or routed to other intracellular organelles. The multivesicular body (MVB) is a late endosome that contains vesicles formed by invagination of the endosomal membrane toward the compartmental lumen. Many growth factor receptors and stimulatory components of the immune system are sorted into MVB vesicles. Furthermore, human immunodeficiency virus-1 and other enveloped RNA viruses usurp components required for MVB vesicle formation in order to produce virions, thereby enabling the spread of viral infection. The objective of this application is to determine the molecular basis of MVB vesicle-mediated transport. Previous work on this project has identified mechanisms by which the Bro1 protein in Saccharomyces cerevisiae participates in the MVB cargo sorting pathway. The proposed work seeks to expand the scope of this analysis to understand the molecular mechanisms that regulate MVB vesicle formation. This process is impaired in cells lacking functional endosomal sorting complexes required for transport (ESCRTs) as a result of endosomes forming `class E compartments.' Preliminary studies suggest that class E compartment formation is driven by the accumulation of ubiquitinated proteins at endosomes and depends on endosomal tethering complexes. Specific aim 1 will determine the mechanism that regulates protein deubiquitination at endosomes, specific aim 2 will explore how ubiquitinated proteins and tethering components cooperate to create class E compartments, and specific aim 3 will investigate the mechanism that regulates polymerization of Snf7, which has been implicated as the driving force of MVB vesicle budding. All three specific aims will be pursued using a combination of genetic, biochemical, and microscopic studies to examine a variety of mutant yeast strains.